Dual Localization Research Articles

Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction.

NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

Differential Expression of Drosophila Transgelins Throughout Development.

Transgelins are a conserved family of actin-binding proteins involved in cytoskeletal remodeling, cell contractility, and cell shape. In both mammals and Drosophila, three genes encode transgelin proteins. Transgelins exhibit a broad and overlapping expression pattern, which has obscured the precise identification of their role in development. Here, we report the first systematic developmental analysis of all Drosophila transgelin proteins, namely, Mp20, CG5023, and Chd64 in the living organism. Drosophila transgelins display overall higher sequence identity with mammalian TAGLN-3 and TAGLN-2 than with TAGLN. Detailed examination in different developmental stages revealed that Mp20 and CG5023 are predominantly expressed in mesodermal tissues with the onset of myogenesis and accumulate in the cytoplasm of all somatic muscles and heart in the late embryo. Notably, at postembryonic developmental stages, Mp20 and CG5023 are detected in the gut’s circumferential muscles with distinct subcellular localization: Z-lines for Mp20 and sarcomere and nucleus for CG5023. Only CG5023 is strongly detected in the adult fly in the abdominal, leg, and synchronous thoracic muscles. Chd64 protein is primarily expressed in endodermal and ectodermal tissues and has a dual subcellular localization in the cytoplasm and the nucleus. During the larval–pupae transition, Chd64 is expressed in the brain, eye, legs, halteres, and wings. In contrast, in the adult fly, Chd64 is expressed in epithelia, including the alimentary tract and genitalia. Based on the non-overlapping tissue expression, we predict that Mp20 and CG5023 mostly cooperate to modulate muscle function, whereas Chd64 has distinct roles in epithelial, neuronal, and endodermal tissues.

Peculiarities of aminoacyl-tRNA synthetases from trypanosomatids

Trypanosomatid parasites are responsible for various human diseases, such as sleeping sickness, animal trypanosomiasis, or cutaneous and visceral leishmaniases. The few available drugs to fight related parasitic infections are often toxic and present poor efficiency and specificity, and thus, finding new molecular targets is imperative. Aminoacyl-tRNA synthetases (aaRSs) are essential components of the translational machinery as they catalyze the specific attachment of an amino acid onto cognate tRNA(s). In trypanosomatids, one gene encodes both cytosolic- and mitochondrial-targeted aaRSs, with only three exceptions. We identify here a unique specific feature of aaRSs from trypanosomatids, which is that most of them harbor distinct insertion and/or extension sequences. Among the 26 identified aaRSs in the trypanosome Leishmania tarentolae, 14 contain an additional domain or a terminal extension, confirmed in mature mRNAs by direct cDNA nanopore sequencing. Moreover, these RNA-Seq data led us to address the question of aaRS dual localization and to determine splice-site locations and the 5′-UTR lengths for each mature aaRS-encoding mRNA. Altogether, our results provided evidence for at least one specific mechanism responsible for mitochondrial addressing of some L. tarentolae aaRSs. We propose that these newly identified features of trypanosomatid aaRSs could be developed as relevant drug targets to combat the diseases caused by these parasites.

Imidazole-Mediated Dual Location Disassembly of Acid-Degradable Intracellular Drug Delivery Block Copolymer Nanoassemblies.

Acid-degradable (or acid-cleavable) polymeric nanoassemblies have witnessed significant progress in anti-cancer drug delivery. However, conventional nanoassemblies designed with acid-cleavable linkages at a single location have several challenges, such as, sluggish degradation, undesired aggregation of degraded products, and difficulty in controlled and on-demand drug release. Herein, a strategy that enables the synthesis of acid-cleavable nanoassemblies labeled with acetaldehyde acetal groups in both hydrophobic cores and at core/corona interfaces, exhibiting synergistic response to acidic pH at dual locations and thus inducing rapid drug release is reported. The systematic analyses suggest that the acid-catalyzed degradation and disassembly are further enhanced by decreasing copolymer concentration (i.e., increasing proton/acetal mole ratio). Moreover, incorporation of acid-ionizable imidazole pendants in the hydrophobic cores improve the encapsulation of doxorubicin, the anticancer drug, through π-π interactions and enhance the acid-catalyzed hydrolysis of acetal linkages situated in the dual locations. Furthermore, the presence of the imidazole pendants induce the occurrence of core-crosslinking that compensates the kinetics of acetal hydrolysis and drug release. These results, combined with in vitro cell toxicity and cellular uptake, suggest the versatility of the dual location acid-degradation strategy in the design and development of effective intracellular drug delivery nanocarriers.

Interconnection of the mycobacterial heparin-binding hemagglutinin with cholesterol degradation and heme/iron pathways identified by proximity-dependent biotin identification in Mycobacterium smegmatis.

Deciphering protein-protein interactions is a critical step in the identification and the understanding of biological mechanisms deployed by pathogenic bacteria. The development of in vivo technologies to characterize these interactions is still in its infancy, especially for bacteria whose subcellular organization is particularly complex, such as mycobacteria. In this work, we used the proximity-dependent biotin identification (BioID) to define the mycobacterial heparin-binding hemagglutinin (HbhA) interactome in the saprophytic bacterium Mycobacterium smegmatis. M. smegmatis is a commonly used model to study and characterize the physiology of pathogenic mycobacteria, such as Mycobacterium tuberculosis. Here, we adapted the BioID technology to in vivo protein-protein interactions studies in M. smegmatis, which presents several advantages, such as maintaining the complex organization of the mycomembrane, offering the possibility to study membrane or cell wall-associated proteins, including HbhA, in the presence of cofactors and post-translational modifications, such as the complex methylation pattern of HbhA. Using this technology, we found that HbhA is interconnected with cholesterol degradation and heme/iron pathways. These results are in line with previous studies showing the dual localization of HbhA, associated with the cell wall and intracytoplasmic lipid inclusions, and its induction under high iron growth conditions.

Impaired phosphatidylethanolamine metabolism activates a reversible stress response that detects and resolves mutant mitochondrial precursors

SummaryPhosphatidylethanolamine (PE) made in mitochondria has long been recognized as an important precursor for phosphatidylcholine production that occurs in the endoplasmic reticulum (ER). Recently, the strict mitochondrial localization of the enzyme that makes PE in the mitochondrion, phosphatidylserine decarboxylase 1 (Psd1), was questioned. Since a dual localization of Psd1 to the ER would have far-reaching implications, we initiated our study to independently re-assess the subcellular distribution of Psd1. Our results support the unavoidable conclusion that the vast majority, if not all, of functional Psd1 resides in the mitochondrion. Through our efforts, we discovered that mutant forms of Psd1 that impair a self-processing step needed for it to become functional are dually localized to the ER when expressed in a PE-limiting environment. We conclude that severely impaired cellular PE metabolism provokes an ER-assisted adaptive response that is capable of identifying and resolving nonfunctional mitochondrial precursors.

Abstract PS1-46: Magnetic seeds to aid targeted axillary dissection

Abstract Introduction: Indications for accurate removal of a previously identified, involved or highly suspicious axillary lymph node include: 1) after neoadjuvant chemotherapy (NACT) to ensure pathological assessment of the correct index node (targeted axillary dissection, TAD) 2) for women with an abnormal/indeterminate node on imaging who may be able to avoid axillary lymph node clearance (ALNC) because they meet Z0011 criteria or may be eligible for POSNOC trial inclusion 3) guided removal of a specific axillary lymph node (eg Rotter’s node recurrence, diagnostic biopsy, inclusion of specific node in ALNC). Dual localisation is either not viable or has an unacceptably high false negative rate in these scenarios and marking of the index node is advised. Although conventional markers are often used prior to NACT, finding that marked node at surgery can pose a challenge. Wire localisation has variable outcomes and scheduling constraints. Magnetic seed localisation, with MagseedTM, allows for easy placement and excision of targeted nodes. Seeds can be accurately placed, do not migrate and can now be placed many months prior to surgery, at the start of NACT. The aim of this study was to report our early experience. Methods: Patients were identified by searching an imaging database for women who had undergone ultrasound (US)-guided MagseedTM insertion into an abnormal axillary lymph node between August 2018 and July 2020. Data on intended use and surgical outcomes were collected retrospectively from electronic patient records (EPR). Patients were categorised into 3 groups as described above. Results: 37 patients were identified, 17 in group 1, 13 in group 2 and 7 in group 3. Only 1 patient had complications with MagseedTM insertion, requiring a second attempt to successfully mark the required node. In 1 patient the MagseedTM was found on the surgical drape, thought to have been lying adjacent to the node intended for excision, this patient was therefore excluded from further evaluation in the results, as were 3 patients with missing data. One patient has yet to come to surgery, all other MagseedsTM have been successfully retrieved. Group 3 indications were disparate, and this group will not be described further. Group 1 - in 10 of 15 patients post NACT the MagseedTM-marked node was found to be the sentinel node by routine mapping techniques (Technetium 99 +/- Patent V blue dye injection), identified as either hot or hot and blue. Of the 15 patients, 6 remained node positive on intra-operative assessment and underwent ALNC whilst 9 (60%) were spared ALNC. 2 patients avoided false negative results as the Magseed™-marked node contained residual disease yet was neither hot nor blue. Group 2 - Ten patients underwent sentinel lymph node biopsy using routine mapping techniques and MagseedTM-guided excision of a specifically targeted node. In 7 the MagseedTM-marked node was found to be a sentinel node (hot or hot and blue). Only 3 patients had sufficient disease to warrant completion ALNC. Conclusion: Insertion of a magnetic seed into a biopsy-proven positive node prior to planned TAD or into an abnormal node identified at diagnosis facilitates specific node retrieval for pathological assessment, with minimal associated complications, and has wide versatility. Magnetic seed localisation reduces the false negative rate and provides confidence in the correct assessment of the axilla thereby sparing patients unnecessary ALNC if they have had a complete pathological response to NACT or have limited axillary disease. The ability to insert the seed prior to the surgical date allows for flexibility with scheduling in comparison to wire localisation. Citation Format: Kathryn E Harborough, V Sinnett, K Downey, K Krupa, J E Rusby, P A Barry. Magnetic seeds to aid targeted axillary dissection [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS1-46.

A New Paradigm in Catalase Research.

Recent findings provide evidence for dynamic and highly regulated dual subcellular localization of catalase, a hydrogen peroxide (H2O2)-metabolizing enzyme, in peroxisomes and the cytosol. These data suggest a number of important implications for the field of oxidative stress biology.

Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization.

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by 1H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

PARTICULAR QUALITIES OF THE PROTEOLYTIC SYSTEM IN PATIENTS WITH TUBERCULOSIS DEPENDING ON THE SENSITIVITY OF THE PATHOGEN

The aim: Studying the features of the proteolytic system in patients with tuberculosis depending on the sensitivity of the pathogen. Materials and methods: In the course of the research we studied the level of elastase in the blood of 111 patients. The first group consisted of 66 (59.5%) people with pulmonary tuberculosis (39 were sensitive to antibacterial drugs, 27 were resistant). The second group included 13 (11.7%) patients with tuberculous pleurisy. The third group consisted of 32 (28.8%) patients with dual localization of the process (pulmonary tuberculosis and pleural tuberculosis). Results: The level of neutrophil elastase in patients with tuberculous pleurisy (253.2 nmol / min • ml) was 2.2 times higher than in patients with sensitive pulmonary tuberculosis (110.1 nmol / min • ml) and higher than in patients with resistant pulmonary tuberculosis 3.0 times. In combined pulmonary and pleural tuberculosis (third group) the level of elastase was 1.6 times higher than in pulmonary tuberculosis (176.9 nmol / min • ml)) (p <0.01), but lower than in pleurisy in 1, 4 times. In sensitive combined tuberculosis (lungs and pleura) the level of NE was 1.5 times higher than in patients of subgroup 1a (p <0.01) and 1.4 times lower than in patients with tuberculous pleurisy (p <0.01 ). Conclusions: The highest level of elastase in tuberculous pleurisy can be explained by its increased production, contributes to increased "permeability" of the pleural sheets and the accumulation of pleural effusion. In resistant forms of tuberculosis, the immune response in the form of the activity of the proteolytic system, which is lower than in sensitive forms, can be explained by the exhaustion of the immune system under the influence of aggressive tuberculosis. The above can be associated with both the weakening of the patient's body and the aggressiveness of the pathogen.

Do we still need to do dual tracer localisation for sentinel node biopsy in patients with early breast cancer?

Introduction: During COVID-19 pandemic the breast cancer services and practice changed significantly to minimise the exposure of patient or staff to COVID-19. The use of a single tracer (isotope) for localisation of sentinel node was suggested rather than gold standard dual tracer localisation by Association of Breast Surgery, UK. March 2020. This was done to avoid the small risk of anaphylaxis associated with blue dye, requiring ventilator support and thus straining the already overwhelmed ITUs.

Impaired Phosphatidylethanolamine Metabolism Activates a Reversible Stress Response that Detects and Resolves Mutant Mitochondrial Precursors

Phosphatidylethanolamine made in mitochondria has long been recognized as an important precursor for phosphatidylcholine production that occurs in the endoplasmic reticulum (ER). Recently, the strict mitochondrial localization of the enzyme that makes PE in the mitochondrion, phosphatidylserine decarboxylase 1 (Psd1), was questioned. Since a dual localization of Psd1 to the ER would have far-reaching implications, we initiated our study to independently re-assess the subcellular distribution of Psd1. Our results support the unavoidable conclusion that the vast majority, if not all, of functional Psd1 resides in the mitochondrion. Through our efforts, we discovered that mutant forms of Psd1 that impair a self-processing step needed for it to become functional are dually localized to the ER when expressed in a PE-limiting environment. We conclude that severely impaired cellular PE metabolism provokes an ER-assisted adaptive response that is capable of identifying and resolving nonfunctional mitochondrial precursors.

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast.

ABSTRACTEukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

Functional characterization of methionine sulfoxide reductases from Leptospira interrogans

BackgroundMethionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. MethodsWe identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. ResultsThe recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. Conclusions and General significanceThis article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.

Dually localised proteins found in both the apicoplast and mitochondrion utilize the Golgi-dependent pathway for apicoplast targeting in Toxoplasma gondii.

Like other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle- the apicoplast. Apicoplast proteins are nuclear encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi-independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, whereas a handful of reports show that a few proteins use the Golgi-dependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known. In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN/IRP) are trafficked through the Golgi, whereas proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast. Collectively, with these results, for the first time, we establish that the driver of the Golgi-dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.

Atomic-orbital-free intrinsic ferromagnetism in electrenes

We report intrinsic ferromagnetism in monolayer electrides or electrenes, in which excess electrons act as anions. Our first-principles calculations demonstrate that magnetism in such electron-rich two-dimensional (2D) materials originates from the anionic electrons rather than partially filled d orbitals, which is fundamentally different from ferromagnetism found in other 2D intrinsic magnetic materials. Taking the honeycomb LaBr$_2$ (La$^{3+}$Br$^{-}_{2}\cdot e^{-}$) as an example, our calculations reveal that the excess electron is localized at the center of the hexagon, which leads to strong Stoner-instability of the associated states at the Fermi energy, resulting in spontaneous magnetization and formation of a local moment. The overlap of extended tails of the wave functions of these electrons mediates a long-range ferromagnetic interaction, contributing to a Curie temperature ($T_\textrm{c}$) of 235 K and a coercive field ($H_\textrm{c}$) of 0.53 T, which can be further enhanced by hole doping. The dual nature, localization and extension, of the electronic states suggests a unique mechanism in such magnetic-element-free electrenes as intrinsic 2D ferromagnets.

Functional Diversification of the Dihydroflavonol 4-Reductase from Camellia nitidissima Chi. in the Control of Polyphenol Biosynthesis.

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. (CnDFR) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR. The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-β-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

The Nuclear Localization of the DnaJ-Like Zinc Finger Domain-Containing Protein EDA3 Affects Seed Development in Arabidopsis thaliana.

The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

Localization and RNA Binding of Mitochondrial Aminoacyl tRNA Synthetases

Mitochondria contain a complete translation machinery that is used to translate its internally transcribed mRNAs. This machinery uses a distinct set of tRNAs that are charged with cognate amino acids inside the organelle. Interestingly, charging is executed by aminoacyl tRNA synthetases (aaRS) that are encoded by the nuclear genome, translated in the cytosol, and need to be imported into the mitochondria. Here, we review import mechanisms of these enzymes with emphasis on those that are localized to both mitochondria and cytosol. Furthermore, we describe RNA recognition features of these enzymes and their interaction with tRNA and non-tRNA molecules. The dual localization of mitochondria-destined aaRSs and their association with various RNA types impose diverse impacts on cellular physiology. Yet, the breadth and significance of these functions are not fully resolved. We highlight here possibilities for future explorations.